one of our users would like to transfer one protein coupled to another one into cells where the coupled protein should be cleaved.
One applicable method is obviously crosslinking the two proteins with a disulfide-containing crosslinker which would be cleaved in the reducing environment of the cytoplasm to release the protein. I wonder if anyone of you knows a protocol where you could use a fusion protein with a protease site. The site could be for TEV or 3C which could be added to the sample or for an endogeneous protease that would cleave the linker and release the target protein.
Thanks in advance!
I once used a system in E. coli for a similar experiment with two plasmids - one with TEV protease under the arabinose promoter, and one with my fusion protein under an IPTG inducible promoter. So in the presence of IPTG and arabinose the fusion protein would get cleaved, but in the absence of arabinose not. This worked nicely, and I'm sure similar systems have been set up for other cell types. If this helps you let me know and I will look up the details, it was about 5 years ago so I don't remember now exactly how I did it.